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Introduction: Extracellular vesicles (EVs) can be released by any cell and are crucial for cell-to-cell communications. EVs have been characterized in patients with solid and hematological tumors, where they play an important role in tumor progression and metastasis. EVs may express different surface proteins derived from the parental cells, including immunomodulatory molecules, such as HLA-G and PDL1., Methods: We isolated EV from bone marrow (BM) samples of patients with Neuroblastoma (NB) and healthy controls and we analyzed the expression of CD56, GD2 and immune checkpoints on EV by flow cytometry. Next, we analyzed the function of T cells in vitro in the presence or absence of NB patients' BM-derived EV, in terms of proliferation and cytokine production. Finally, we analyzed the correlation between the expression of immune checkpoints on EV and the clinical outcome of patients., Results: We found a higher expression of CD56 on EVs derived from BM of patients with NB than in those from healthy donors (HD). However, CD56 expression was not dependent on BM infiltration of NB cells. Moreover, the analysis of GD2 expression revealed that only a small fraction of EVs was released by infiltrating NB cells, whereas the majority may derive from BM-resident cells. BM-derived EVs from NB patients display a higher expression of HLA-G and PD-L1 than those derived from HD. Nonetheless, such EVs are able to modulate T cell immune responses. We measured a robust response, in vitro, towards a common bacterial antigen, including the release of GM-CSF and proinflammatory cytokines, like IFN-a and IL-6, from mononuclear cells. Some of these immunomodulatory features are dependent on the expression of HLA-G and PD-L1, whereas others may rely on other mechanism(s). Finally, a high expression of CD56, HLA-G and PD-L1 on BM-derived EVs may represent a good prognostic factor., Conclusions: We described the presence of HLA-G and PDL1-bearing EVs in the BM of NB patients, which may represent a mechanism performed by resident BM cells to counteract the inflammation occurring in the BM microenvironment of NB patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Marimpietri, Corrias, Tripodi, Gramignoli, Airoldi and Morandi.)

Radiofrequency ablation (RFA) under endoscopic ultrasound (EUS) guidance has been developed and utilized over the last decade to provide the loco-regional treatment of solid and cystic pancreatic neoplastic lesions. The advantage of this approach relies on the close proximity of the EUS transducer to the target pancreatic lesion, which, coupled with the development of specifically designed RFA ablation devices, has made the procedure minimally invasive, with a clear reduction in adverse events as compared to the high morbidity of the surgical approach. EUS-RFA has been applied so far to pancreatic functional and non-functional neuroendocrine neoplasms, pancreatic ductal adenocarcinoma or metastases to the pancreas, and pancreatic neoplastic cysts. Excluding neuroendocrine tumors, for other indications, most of these procedures have been performed in patients who refused surgery or were at high surgical risk. More studies evaluating EUS-RFA in selected patients, not at surgical risk, are gradually becoming available and will pave the road to extend the indications for this therapeutic approach, also in association with other oncological therapies. The present manuscript will critically review the available evidence in the field of the EUS-guided RFA of solid and cystic pancreatic neoplasms.

Competing Interests: Dr Murina is a speaker for Abbvie, Amgen, Bristol-Meyers-Squibb, and Janssen. She has served as a consultant for Bristol-Meyers-Squibb, Janssen, Novartis, Ortho-Dermatologics, Takeda, and UCB. She is on the Editorial Board of the JAADCR. Drs Gowda, Yang, Tripodi, and Ragland have no conflicts of interest to declare.

Aqueous extracts from four different American hops varieties (Mosaic, Chinook, Citra, and Centennial) were produced using the Synergy Pure technique, an innovative extraction distillation process developed by Synergy Flavours, a global specialist in the manufacturing of flavors, extracts and essences. This process is able to preserve and maximize the aromatic characteristics without increasing the bitterness of the final product. Therefore, the aim of this work is to identify the volatile and sensory characteristics of these extracts, with the additional aim to assess their suitability for brewing. GC-MS and sensory analyses were carried out on the four different aqueous extracts. The results highlighted the presence of 33 volatile compounds in a quantity that allowed us to identify the characteristics of the varieties under investigation in each extract. Sensory analysis was carried out using the expert sensory profiling technique. The results regarding the olfactory analysis of the extracts allowed us to define the aroma profiles of the four extracts, highlighting their strong correspondence with the characteristic of the varieties under investigation. Finally, the results showed that the aqueous extracts produced using the Synergy Pure extraction technique had a richer aroma profile, highlighting its higher aptitude in beer production.

Introduction: Quality and safety of a cell product, essential to guarantee the health of patients, depends on many factors including an appropriate environmental monitoring of the manufacturing rooms. Nonetheless, the maintenance of a controlled environment is requested to minimize the risk of contamination. Thus, a timely detection of changes in microbiological trends is important to adopt promptly effective measures against resistant strains that, in turn, may invalidate not only the sanitization procedures but also the safety of the cell product., Methods: We analyzed microbes found in our cell processing clean room over the last 5 years. We used 10.147 plates for air sampler, passive air monitoring and for checking instruments and operators of the production unit., Results: From these plates, 747 colonies were subjected to identification by the MALDI-TOF Vitek® MS system and the large majority of them was gram positive (97.8%) as witnessed by the finding that the most represented genera harvested from the classified areas were Staphylococcus (65%), Micrococcus (13%), Kocuria (8%) and Bacillus (5%). We never detected fungi. Most microbes found in the operators (both from class A and B) were collected from forearms and resulted of the Staphylococcus genus., Conclusions: The observed microbial contamination is to be attributed to the personnel and no substantial microbial pitfalls in our Cell Factory has been detected., Competing Interests: The authors of this manuscript declare no conflict of interest., (© 2024 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

Introduction: Ground level falls in the elderly often lead to complications due to use of anticoagulants (ACs). Intracranial hemorrhage (ICH), immediate or delayed, is a feared consequence of such falls. The rate of delayed ICH (dICH) in patients taking anticoagulants or antiplatelet (AP) agents ranges from .6% to 6%. Patients on warfarin have a persistent rate of dICH, leading to implementation of routine repeat head CTs at our institution. This policy was extended to direct oral anticoagulants (DOACs). This study aims to determine institutional incidence of DOAC-associated dICH., Methods: With IRB waiver approval, we conducted a retrospective review of trauma evaluations for falls on DOACs from 2016 to 2018. We reviewed records for neurologic status, DOAC use, and results of initial and delayed head CTs. Exclusion criteria included initial GCS ≤14, new neurologic deficits, traumatic findings on initial CT, concurrent use of additional AC/AP, or absence of repeat head CT., Results: Among 632 patients evaluated for falls on AC/AP therapy, 159 (25%) of patients were included in the review. The age range was 19-98 years old, with 99 females and 60 males. Half of the patients were on apixaban, with the rest on dabigatran or rivaroxaban. Ten patients presented with GCS of 14. No delayed hemorrhages were detected in this population., Conclusion: The necessity of a repeat head CT in patients taking DOACs is debated in the literature. Our analysis failed to demonstrate any delayed hemorrhage in neurologically intact patients after head strike on DOAC, suggesting no indication for follow-up imaging in this group., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Endoscopic ultrasound (EUS) guided biliary drainage (BD) is an accepted salvage procedure in patients with distal malignant biliary obstruction (DMBO) when endoscopic retrograde cholangiopancreatography (ERCP) is unsuccessful. The potential advantages of EUS-BD include gastric or duodenal biliary access, utilization of novel biliary stents and stent placement away from the area of stenosis, resulting in longer stent patency. These features make EUS-BD very appealing as a primary procedure for biliary drainage. There is a growing body of evidence supporting the utilization of EUS as a primary drainage procedure instead of ERCP, with comparable outcomes.

Competing Interests: The authors declare that they have no conflict of interest.

The aim of the study was to try to obtain more information on diagnostic samplings and biomarkers using dual-layer spectral CT in lung biopsies. Lung biopsies were performed by merging images obtained with CBCT with those from spectral CT to use them as functional guidance, experimenting with double sampling to determine the difference between the area with a higher Z-effective number and that with a lower Z-effective number. Ten patients with large lung lesions on spectral CT were selected and underwent percutaneous transthoracic lung mass biopsy. Technical success was calculated. The percentage of neoplastic, inflammatory, fibrotic, necrotic cells, or non-neoplastic lung parenchyma was reported. The possibility of carrying out immunohistochemical or molecular biology investigations was analyzed. All lesions were results malignant in 10/10 samples in the Z max areas; in the Z min areas, malignant cells were found in 7/10 samples. Technical success was achieved in 100% of cases for Z max sampling and in 70% for Z min sampling ( p -value: 0.2105). The biomolecular profile was detected in 9/10 (90%) cases in Z max areas, while in 4/10 (40%) cases in Z min areas ( p -value: 0.0573). The advantage of Z-effective imaging would be to identify a region of the lesion that is highly vascularized and probably richer in neoplastic cells, thus decreasing the risk of obtaining a non-diagnostic biopsy sample.

Endoscopic ultrasound-guided gallbladder drainage (EUS-GBD) has emerged over the last years as an alternative procedure to percutaneous drainage (PT)-GBD in patients with acute cholecystitis (AC) at high surgical risk. This process has been driven by the advent of lumen-apposing metal stents (LAMS) with electrocautery-enhanced capability, which has rendered the drainage procedure easier to accomplish and safer. Studies and meta-analyses have proven the superiority of EUS-GBD over PT-GBD in high-surgical-risk patients with AC. Little evidence exists in the same setting that EUS-GBD compares equally with laparoscopic cholecystectomy (LC). Moreover, EUS-GBD might theoretically have a possible role in patients at high surgical risk with an indication to undergo cholecystectomy or with a high probability of conversion from LC to open cholecystectomy. Properly designed studies are needed to better clarify the role of EUS-GBD in these patient populations., (© 2023 The Authors. Digestive Endoscopy published by John Wiley & Sons Australia, Ltd on behalf of Japan Gastroenterological Endoscopy Society.)

Gastric outlet obstruction (GOO) is a clinical syndrome traditionally managed by surgical gastrojejunostomy or enteral stenting. The surgical approach is burdened with a high rate of adverse events (AEs), while enteral stenting has a limited long-term clinical effectiveness, with the need for repeat procedures. The availability of lumen-apposing metal stents (LAMSs) has resulted a shift in the treatment paradigm of GOO. Indeed, endoscopists are now able to create a stable anastomosis between the stomach and small bowel under endosonographic guidance. EUS-guided gastro-enteroanastomosis (EUS-GE) has the theoretical advantage of a durable luminal patency resulting from stent placement away from the site of obstruction, free from surgical-related AEs. This approach could be especially valuable in terminally ill patients with a limited life expectancy. The present paper reviews procedural techniques and clinical outcomes of EUS-GE in the context of both malignant and benign GOOs.

Solid pancreatic neoplasms are one of the most diagnosed gastrointestinal malignancies thanks to the current and progressive advances in radiologic methods. Endoscopic ultrasound-guided techniques have over time gained a prominent role in the differential diagnosis and characterization of these pancreatic lesions, including pancreatic cancer, neuroendocrine tumors, and metastases. Recently, several endoscopic ultrasound-guided locoregional treatment techniques, which are divided into thermal ablative techniques and non-thermal injection techniques, have been developed and applied in different settings for the treatment of solid pancreatic neoplasms. The most common ablative techniques are radiofrequency, microwave, laser, photodynamic therapy and hybrid techniques such as hybrid cryothermal ablation. The most common injection techniques are ethanol injection, immunotherapy and brachytherapy. In this review, we update evidence about the efficacy and safety of endoscopic ultrasound-guided locoregional treatments for solid pancreatic neoplasms.

Hemp seed flour (HSF) is a by-product of the hemp oil production process and is a valuable source of protein, dietary fiber, minerals, and vitamins. In line with sustainable food production and a circular economy, this research aimed to utilize HSF as fortifying ingredient in the production of gnocchi , a typical Italian potato-based fresh pasta, and to investigate the effects of the addition on the quality and consumers' acceptability of the enriched products. Three formulations have been developed using 5-20% HSF in substitution of soft wheat flour. Nutritional value, cooking quality, color, texture and sensory profile, and the consumers' acceptability of gnocchi samples were evaluated, as well as the functional properties of pure and composite flours and the HSF aroma compounds. HSF addition allowed to enhance the nutritional value of gnocchi , gaining the nutritional claim of "source of fiber" in case of formulations with ≥10% of HSF. Moreover, the fortified gnocchi had a high technological quality in terms of cooking loss, cooking resistance and textural properties, and average sensory quality; however, the vegetable and hemp odor and the bitter taste make them not well appreciated by consumers highlighting the need for improving the HSF sensory quality for consumers' satisfaction.

Neuroblastoma (NB) is the most common extracranial malignant tumor in children. Although the survival rate of NB has improved over the years, the outcome of NB still remains poor for over 30% of cases. A more accurate risk stratification remains a key point in the study of NB and the availability of novel prognostic biomarkers of "high-risk" at diagnosis could help improving patient stratification and predicting outcome. In this paper we show a biomarker discovery approach applied to the plasma of 172 NB patients. Plasma samples from a first cohort of NB patients and age-matched healthy controls were used for untargeted metabolomics analysis based on high-resolution mass spectrometry (HRMS). Differential expression analysis highlighted a number of metabolites annotated with a high degree of identification. Among them, 3-O-methyldopa (3-O-MD) was validated in a second cohort of NB patients using a targeted metabolite profiling approach and its prognostic potential was also analyzed by survival analysis on patients with 3 years follow-up. High expression of 3-O-MD was associated with worse prognosis in the subset of patients with stage M tumor (log-rank p < 0.05) and, among them, it was confirmed as a prognostic factor able to stratify high-risk patients older than 18 months. 3-O-MD might be thus considered as a novel prognostic biomarker of NB eligible to be included at diagnosis among catecholamine metabolite panels in prospective clinical studies. Further studies are warranted to exploit other potential biomarkers highlighted using our approach., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Barco, Lavarello, Cangelosi, Morini, Eva, Oneto, Uva, Tripodi, Garaventa, Conte, Petretto and Cangemi.)

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC-negative cases or SNPM with histological assessment for MCC-positive cases. A result was obtained in 93% (29/31) of cases with a diagnostic yield of 45.1% (14/31) with the proposed workflow, compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

Cu I /TEMPO (TEMPO=2,2,6,6-tetramethylpiperidinyloxyl) catalyst systems are versatile catalysts for aerobic alcohol oxidation reactions to selectively yield aldehydes. However, several aspects of the mechanism are yet unresolved, mainly because of the lack of identification of any reactive intermediates. Herein, we report the synthesis and characterization of a dinuclear [L1 2 Cu 2 ] 2+ complex 1, which in presence of TEMPO can couple the catalytic 4 H + /4 e - reduction of O 2 to water to the oxidation of benzylic and aliphatic alcohols. The mechanisms of the O 2 -reduction and alcohol oxidation reactions have been clarified by the spectroscopic detection of the reactive intermediates in the gas and condensed phases, as well as by kinetic studies on each step in the catalytic cycles. Bis(μ-oxo)dicopper(III) (2) and bis(μ-hydroxo)dicopper(II) species 3 are shown as viable reactants in oxidation catalysis. The present study provides deep mechanistic insight into the aerobic oxidation of alcohols that should serve as a valuable foundation for ongoing efforts dedicated towards the understanding of transition-metal catalysts involving redox-active organic cocatalysts., (© 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

The role of therapeutic drug monitoring (TDM) of valaciclovir (VA)/aciclovir (A) and valganciclovir/ganciclovir (VG/G) in critically ill patients is still a matter of debate. More data on the dose-concentration relationship might therefore be useful, especially in pediatrics where clinical practice is not adequately supported by robust PK studies. We developed and validated a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) micro-method to simultaneously quantify A and G from plasma and dried plasma spots (DPS). The method was based on rapid organic extraction from DPS and separation on a reversed-phase C-18 UHPLC column after addition of deuterated internal standards. Accurate analyte quantification using SRM detection was then obtained using a Thermo Fisher Quantiva triple-quadrupole MS coupled to an Ultimate 3000 UHPLC. It was validated following international (EMA) guidelines for bioanalytical method validation and was tested on samples from pediatric patients treated with A, VG, or G for cytomegalovirus infection following solid organ or hematopoietic stem cell transplantation. Concentrations obtained from plasma and DPS were compared using Passing-Bablok and Bland-Altman statistical tests. The assay was linear over wide concentration ranges (0.01-20 mg/L) in both plasma and DPS for A and G, suitable for the expected therapeutic ranges for both Cmin and Cmax, accurate, and reproducible in the absence of matrix effects. The results obtained from plasma and DPS were comparable. Using an LC-MS/MS method allowed us to obtain a very specific, sensitive, and rapid quantification of these antiviral drugs starting from very low volumes (50 μL) of plasma samples and DPS. The stability of analytes for at least 30 days allows for cost-effective shipment and storage at room temperature. Our method is suitable for TDM and could be helpful for improving knowledge on PK/PD targets of antivirals in critically ill pediatric patients.

Background: As part of the fight against SARS CoV2 infection, vaccination program for health workers at Giannina Gaslini pediatric hospital (IGG) in Genoa, Italy, started on December 2020. We evaluated the anti-Spike protein response in healthcare workers after a complete vaccination scheme of 2 doses spaced by 3 weeks., Methods: Immunoglobulin class G (IgG) against SARS-CoV-2 spike RBD were detected by means of a chemiluminescence immunoassay for quantitative IgG antibodies using Maglumi SARS-CoV-2-S-RBD IgG kit during the 3rd week after vaccination completion., Results: IgG anti SARS-CoV-2 spike protein were detected in 99.88% of 1765 healthcare workers 3 weeks after 2nd dose of BNT162b2. Higher median IgG values were observed in younger subjects (807 UA/mL in under 30 vs 429 UA/mL in over 60; p < 0.001) and those with previous COVID-19 (1284 vs 574 UA/mL; p < 0.001)., Conclusion: BNT162b2 is effective in inducing anti SARS-CoV-2 antibodies even in real-life setting. The higher antibody title observed in workers with a previous documented SARS CoV2 infection confirms the possibility to carry out only one dose of BNT162b2 in a context of vaccines shortage., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Adenosine Deaminase 2 Deficiency (DADA2) (OMIM: 607575) is a monogenic, autoinflammatory disease caused by the loss of functional homozygous or heterozygous mutations in the ADA 2 gene (previously CECR1, Cat Eye Syndrome Chromosome Region 1). A timely diagnosis is crucial to start Anti-TNF therapies that are efficacious in controlling the disease. The confirmation of DADA2 is based on DNA sequencing and enzymatic assay. It is, thus, very important to have robust and reliable assays that can be rapidly utilized in specialized laboratories that can centralize samples from other centers. In this paper, we show a novel enzymatic assay based on liquid chromatography-tandem mass spectrometry that allows the accurate determination of the ADA2 enzyme activity starting from very small amounts of plasma spotted on filter paper (dried plasma spot). The method allows significantly distinguishing healthy controls from affected patients and carriers and could be of help in implementing the diagnostic workflow of DADA2.

Liquid-chromatography coupled to high resolution mass spectrometry (LC-HRMS) is currently the method of choice for untargeted metabolomic analysis. The availability of established protocols to achieve a high confidence identification of metabolites is crucial. The aim of this work is to describe the workflow that we have applied to build an Accurate Mass Retention Time (AMRT) database using a commercial metabolite library of standards. LC-HRMS analysis was carried out using a Vanquish Horizon UHPLC system coupled to a Q-Exactive Plus Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientific, Milan, Italy). The fragmentation spectra, obtained with 12 collision energies, were acquired for each metabolite, in both polarities, through flow injection analysis. Several chromatographic conditions were tested to obtain a protocol that yielded stable retention times. The adopted chromatographic protocol included a gradient separation using a reversed phase (Waters Acquity BEH C18) and a HILIC (Waters Acquity BEH Amide) column. An AMRT database of 518 compounds was obtained and tested on real plasma and urine samples analyzed in data-dependent acquisition mode. Our AMRT library allowed a level 1 identification, according to the Metabolomics Standards Initiative, of 132 and 124 metabolites in human pediatric plasma and urine samples, respectively. This library represents a starting point for future metabolomic studies in pediatric settings.

Medical cannabis is increasingly being used in the treatment and support of several diseases and syndromes. The quantitative determination of active ingredients (delta-9 tetrahydrocannabinol, THC, and cannabidiol, CBD) in galenic oily preparations is prescribed by law for each produced batch. The aim of this work is to describe the organization of the titration activity centralized at three regional reference laboratories in Northern Italy. Pre-analytical, analytical, and post-analytical phases have been defined in order to guarantee high quality standards. A cross-validation between laboratories allowed for the definition of the procedures that guarantee the interchangeability between reference laboratories. The risk management protocol adopted can be useful for others who need to undertake this activity.

Purpose: Interest in cannabis-based therapies has recently increased, due to the availability of cannabidiol (CBD) for the treatment of epilepsy without psychoactive effects. Therapeutic drug monitoring can prevent drug interactions and minimize drug toxicity. We evaluated a volumetric absorptive microsampling (VAMS) method combined with LC-MS/MS (liquid chromatography coupled to tandem mass spectrometry) for the quantification of CBD blood levels in patients with refractory epilepsy. Methods: Prospective observation of patients with Dravet syndrome receiving open-label, add-on GW-purified CBD (Epidyolex ® ) at different doses. CBD plasma samples were obtained from venipuncture and LC-MS/MS was used to measure CBD in venous and capillary blood samples collected by VAMS. Results: We enrolled five patients with a mean age of 13 (range: 4-27) years. CBD levels measured by VAMS on capillary blood did not differ from CBD levels measured in plasma by venipuncture ( R 2 > 0.93). Conclusion: This proof-of-concept study suggests that VAMS allows monitoring of CBD plasma levels and can offer valuable support for personalized therapy in refractory epilepsy., (Copyright © 2020 Dubois, Marchese, Pigliasco, Barco, Tripodi, Lomonaco, Lattanzi, Russo, Cangemi and Striano.)

Scientific discovery is shaped by scientists' choices and thus by their career patterns. The increasing knowledge required to work at the frontier of science makes it harder for an individual to embark on unexplored paths. Yet collaborations can reduce learning costs-albeit at the expense of increased coordination costs. In this article, we use data on the publication histories of a very large sample of physicists to measure the effects of knowledge and social relatedness on their diversification strategies. Using bipartite networks, we compute a measure of topic similarity and a measure of social proximity. We find that scientists' strategies are not random, and that they are significantly affected by both. Knowledge relatedness across topics explains [Formula: see text] of logistic regression deviances and social relatedness as much as [Formula: see text], suggesting that science is an eminently social enterprise: when scientists move out of their core specialization, they do so through collaborations. Interestingly, we also find a significant negative interaction between knowledge and social relatedness, suggesting that the farther scientists move from their specialization, the more they rely on collaborations. Our results provide a starting point for broader quantitative analyses of scientific diversification strategies, which could also be extended to the domain of technological innovation-offering insights from a comparative and policy perspective.

The aim of this work is to evaluate volumetric absorptive microsampling (VAMS) from capillary blood as an alternative strategy for therapeutic drug monitoring (TDM) in patients treated with the newly available GW-purified form of cannabidiol (Epidiolex ® ). A fast ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) coupled to an online sample preparation system analysis was carried out on a Thermo Scientific Ultimate 3000 LC system coupled to a TSQ Quantiva triple quadrupole for the quantification of cannabidiol (CBD) and, in addition, delta-9-tetrahydrocannabinol (Δ9-THC). After validation using European Medicine Agency (EMA) guidelines the method was applied to samples obtained by finger prick of five pediatric patients treated with Epidiolex ® and the results were compared to those obtained from venous blood and plasma. The method is linear in the range of 1-800 µg/L for both CBD and THC with intra- and inter-day precisions ranging from 5% to 14% and accuracies from -13% to +14% starting from 30 µL of sample. Stability in VAMS is ensured for up to 4 weeks at 25 °C thus allowing simple delivery. There was no difference ( p = 0.69) between concentrations of CBD measured from VAMS sampled from capillary or venous blood (range: 52.19-330.14 or 72.15-383.45 µg/L) and those obtained from plasma (range: 64.3-374.09 µg/L) The VAMS-LC-MS/MS method represents a valid alternative strategy for therapeutic drug monitoring of patients treated with Epidiolex ® .

Background: The accurate measurement of plasma levels of antibiotics is crucial for the individualization of antimicrobial therapies based on PK/PD strategies. In this paper we describe a new rapid and simple LC-MS/MS platform for quantifying 14 antibiotics (amikacin, amoxicillin, ceftazidime, ciprofloxacin, colistin, daptomycin, gentamicin, linezolid, meropenem, piperacillin, teicoplanin, tigecycline, tobramycin and vancomycin) and a beta-lactamase inhibitor (tazobactam) starting from 50 μL plasma samples., Methods: Analyses were performed on a Thermo Scientific™ Ultimate™ 3000 LC system (Thermo Fisher Scientific, Milan, Italy) coupled to a Thermo Scientific™ TSQ Quantiva™ Triple Quadrupole mass spectrometer. After fast protein precipitation protocols and addition of deuterated internal standards, samples were subjected to a fast HPLC gradient separation and the 15 drugs were quantified using multiple reaction monitoring of specific transitions over a wide range of concentrations. The suitability of the assay for TDM was tested on plasma samples derived from pediatric patients under treatment with one or more antibiotics., Results: The overall turnaround time of the assay was 20 min. The assay was validated following EMA guidelines for bioanalytical method validation and showed excellent accuracy (ranging from 85.3 and 112.7) and reproducibility (ranging from 1.3 to 9.7) as well as the absence of matrix effects (<15 %) for all the drugs tested. The lower limits of quantifications were between 0.1 and 2 mg/L. the recovery rate exceeded 85 % for all the drug tested. Stability was evaluated in different conditions thus allowing the setting up of reliable operative procedures., Conclusions: This work provides a LC-MS/MS platform validated for clinical use for a rapid quantification of a broad spectrum of drugs having different chemical characteristics in a small volume of plasma and is suitable for real-time TDM-guided personalization of antimicrobial treatment in critically ill patients., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: Newborns, admitted to the Neonatal Intensive Care Unit (NICU), are exposed to a large number of medications, the majority of which are not labeled for use in infants, especially in preterm newborns, because clinical trials on their benefits and harms are lacking. There is a huge gap in knowledge on pharmacokinetic (PK) data in sick preterm infants, including that of drug penetration to cerebrospinal fluid (CSF). One of the issues is related to the lack of reliable analytical methods for the measurement of drugs in CSF., Methods: In this paper we describe a specific and sensitive LC-MS/MS method for the simultaneous quantification in CSF of four commonly prescribed drugs in NICUs: caffeine, betamethasone, clonidine and furosemide., Results: The method was validated following EMA guidelines and applied to several CSF samples of preterm infants with post-hemorrhagic ventricular dilatation in which a ventricular access device was applied. The range of the concentrations of the four drugs measured in the CSF was wide., Conclusions: Our method can be considered useful for further clinical studies to describe the PK aspects of these drugs in neonatal medicine., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: In recent years, mini-watermelons have increased in popularity. To maintain production and quality standards, various agronomic techniques have been applied. For the Cucurbitaceae family, grafting technique has been used to improve resistance to abiotic stresses, crop productivity and fruit qualitative characteristics. There is some previous literature on this matter, but no information on the influence of grafting on the aroma compounds of mini-watermelons is available. Hence, our research aimed to evaluate the effect of some rootstocks, which were selected on the basis of their tolerance to pathogens, on the quality of mini-watermelons, with particular attention to the volatile aroma compounds., Results: Volatile aroma compounds were analysed using solid-phase microextraction-gas chromatography-mass spectrometry. The most represented compounds were C 6 and C 9 aldehydes and alcohols, which characterize the fruit aroma of the Cucurbitaceae family: (Z)-2-nonenal, (E,Z)-2,6-nonadienal, (Z)-3-nonen-1-ol and (Z,Z)-3,6-nonadien-1-ol were prevalent. Quantitative differences resulted in relation to the various selected rootstocks. Among these, the RS841 rootstock was found to be the most suitable for maintaining yield, quality parameters, sensory characteristics and volatile aroma compounds of mini-watermelon fruits., Conclusions: The results highlighted that the rootstock, as for other Cucurbitaceae varieties, influences fruit quality and plant yield also for the mini-water melon; the results show the importance of screening for rootstock/scion combinations in order to select a graft able to provide resistance to abiotic stresses, and at the same time improve yield and fruit quality. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

There is evidence that chronic hepatitis B virus (HBV) infection is associated with an increased risk of intrahepatic cholangiocarcinoma (ICC) development, and it has been hypothesized an etiological role of HBV in the development of this tumor. Very little is known about occult HBV infection (OBI) in ICC. Aims of the study were to investigate the OBI prevalence and to characterize the HBV molecular status at intrahepatic level in OBI-positive cases with ICC. Frozen liver tumor specimens from 47 HBV surface-antigen-negative patients with ICC and 41 paired non-tumor liver tissues were tested for OBI by 4 different HBV-specific nested PCR. Covalently closed circular HBV DNA (HBV cccDNA) and viral integrations were investigated in OBI-positive cases. HBV DNA was detected in tumor and/or non-tumor specimens from 29/47 (61.7%) ICC patients. HBV cccDNA was found in tissues from 5/17 (34.5%) cases examined. HBV integration was detected in 4/10 (40%) tumor tissues tested and involved HBx and HBV-core gene sequences in 3 and 1 cases, respectively. Viral integration occurred: (a) 9,367 nucleotides upstream of the cat-eye-syndrome critical region protein-5-isoform coding sequence; (b) within the cystinosin isoform-1-precursor gene; (c) within the thromboxane-A-synthase-1 gene; (d) within the ATPase phospholipid transporting 9B gene. Occult HBV infection is highly prevalent in patients with ICC. Both free viral genomes and integrated HBV DNA can be present in these cases. These results suggest an involvement of HBV in the carcinogenic process leading to ICC development even in cases with occult infection., Competing Interests: CONFLICTS OF INTEREST The authors declare no conflicts of interest.

Starting from a warning of possible artificially high plasma colistin concentrations observed in clinical samples in our recently published LC-MS/MS method, we reviewed our analytical assay and demonstrated, for the first time, that caution must be paid to ionization conditions employed in the MS system. The artefact reported here demonstrates that additional experiments should be performed in the course of validation of LC-MS/MS methods to be used in samples containing both colistin and colistimethate., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Introduction: A substantial number of patients with neuroblastoma (NB) have increased excretion of catecholamines and metanephrines. Here, we have investigated the diagnostic role of plasma free metanephrines (PFM), metanephrine (MN), normetanephrine (NMN) and 3-methoxytyramine (3MT) for NB, the most common extra-cranial solid tumour in children., Methods: PFM were quantified by using a commercial IVD-CE LC-MS/MS method on a TSQ Quantiva coupled to an Ultimate 3000. The method was further validated on 103 samples from pediatric subjects (54 patients with histologically confirmed NB and 49 age and sex matched controls). Correlations between PFM concentrations with clinical factors were tested. We directly compared MN, NMN, and 3MT concentrations in matched plasma and urine samples of NB patients (n = 29)., Results: 3MT and NMN showed an excellent diagnostic performance with very high specificity (100% and 95.8%, respectively) and sensitivity (88.2% and 80.4%). ROC curves were obtained (AUC of 0.93 and 0.91 for 3MT and NMN, respectively) and optimal cut-offs that could discriminate between controls and NB patients were defined. A positive correlation between NMN levels in urine and plasma (p = .0017) was found., Discussion: The determination of plasma 3MT and NMN should be taken in consideration as a new diagnostic tool for NB. Validation in prospective clinical studies in comparison to urinary catecholamines and metanephrines is warranted., (Copyright © 2019 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Background: There is controversial data on possible occult HBV reactivation in HCV patients successfully treated with direct-acting antivirals (DAA). However, diagnosis of occult HBV infection (OBI) was not performed by gold standard procedures in any study., Methods: By using several highly sensitive assays, we examined serially collected serum samples from 40 HBV-surface-negative DAA-treated HCV patients with OBI identified by testing liver biopsy specimens through nested-PCR technique. Serum samples were obtained at four time points from each patient (at baseline, at 4 weeks after starting, at the end and 12 weeks after stopping therapy) and tested for HBV DNA by nested-PCR and real-time PCR techniques., Results: All tested serum samples were negative by both quantitative HBV surface antigen (HBsAg) and HBV core-related antigen assays. 26/40 patients were anti-HBs-positive and in all of them, the amount of this antibody was stable at the four time points evaluated. Serum HBV DNA was detected in 10 samples at baseline, in 6 samples 4 weeks after starting therapy, in 11 samples at the end of therapy and in 21 samples 12 weeks after stopping treatment (P=0.001). Aminotransferase values dropped within the normal levels at week 4 of therapy and persisted normal over time in all cases., Conclusions: A slight increase in the amount of HBV DNA 3 months after stopping DAA therapy was the only parameter showing a possible reappearance of HBV activity in OBI patients cured for a concomitant HCV infection, but it was insufficient to lead toward a virological reactivation capable of inducing liver injury.

Aim: Monitoring of blood levels of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) is necessary for optimization of administration of medical cannabis. We describe the validation of a ultra-HPLC-MS/MS method for quantifying THC and CBD from plasma and decoctions and its application for therapeutic drug monitoring. Materials & methods: Analyses were performed by using a TSQ Quantiva™ Triple Quadrupole coupled to a Ultimate 3000 UHPLC system with atmospheric pressure chemical ionization after sample preparation with a straightforward method with deuterated internal standards. Results:  The method has been validated following EMA guidelines and is linear in plasma from 0.16 to 10 ng/ml for both THC and CBD and in decoctions from 4.7 to 600 ng/ml. Conclusion: Given the unpredictable pharmacokinetic behavior of THC and CBD in patients, monitoring of plasma concentrations is strongly recommended for patients under treatment with medical cannabis.

Hepatitis B virus (HBV) infection is the main risk factor for hepatocellular carcinoma (HCC) worldwide. Integration of HBV DNA into the human genome has been found in >80% of HBV-related HCC cases. Some studies have, however, found similar integration patterns in tumorous and nontumorous tissues. Thus, the role of integrations for the development of HCC as well as the rate of integration in different stages of infection remain unclear. The aim of this study was to investigate integrations in patients without HCC, representing different stages of chronic HBV (CHB) infection. Extracted DNA in liver biopsies from 74 patients (one with 2 available biopsies) with CHB infection was analyzed by Alu-PCR. Amplicons were further analyzed by Sanger sequencing. Integration was detected in 39 biopsies (52%) as an amplicon containing both human and HBV sequences by Alu-PCR with one primer targeting a region in the HBV genome. Integrations were found in patients representing the different stages of CHB infection. A majority of the HBV sequences were located upstream or downstream of nucleotide position 1820, which previously has been identified as a common breakpoint in the HBV genome in integrated sequences. Approximately 60% of the HBV integrations were found in noncoding regions of the human genome. Integrations of HBV DNA into the human genome is an event frequently found in mild phases of chronic hepatitis., (© 2018 Wiley Periodicals, Inc.)

Background: Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder caused by sporadic heterozygous mutations in ACVR1 gene which progressively leads to severe heterotopic ossification. FOP is characterized by episodic flare-ups triggered by different factors such as viral infections, tissue injuries, vaccinations, or occurring without a recognizable cause. The sporadic course of the disease, the documented presence of an important inflammatory reaction in early lesions and the partial response to corticosteroids support the idea that the immune system, and in particular the innate component, may play a role in FOP pathogenesis. However, an extensive expression profile of the peripheral blood mononuclear cells (PBMC) of FOP patients has never been done., Methods: In this study, we carried out a wide PBMC immunophenotyping on a cohort of FOP patients and matching controls by multiparametric analysis of the expression of a panel of 37 markers associated with migration, adhesion, inhibition, activation, and cell death of circulating immune cells., Results: We observed a statistically significant increase of the expression of DNAM1 receptor in patients' monocytes as compared to controls, and little but significant differences in the expression profile of CXCR1 (CD181), CD62L, CXCR4 (CD184), and HLA-DR molecules., Conclusions: DNAM1 had been previously shown to play a pivotal role in monocyte migration through the endothelial barrier and the increased expression detected in patients' monocytes might suggest a role of this surface receptor during the early phases of FOP flare-ups in which the activation of the immune response is believed to represent a crucial event. © 2017 International Clinical Cytometry Society., (© 2017 International Clinical Cytometry Society.)

Motivated behaviors often are characterized by a high degree of behavioral activation and work output, and organisms frequently make effort-related decisions based upon cost/benefit analyses. Moreover, people with depression and other disorders frequently show effort-related motivational symptoms, such as anergia, psychomotor retardation, and fatigue. Tasks measuring effort-related choice are being used as animal models of these motivational symptoms. The present studies characterized the ability of the monoamine oxidase -B (MAO-B) inhibitor deprenyl (selegiline) to enhance selection of high-effort lever pressing in rats tested on a concurrent progressive ratio (PROG)/chow feeding choice task. Deprenyl is widely used as an antiparkinsonian drug, but it also has been shown to have antidepressant effects in humans, and to induce antidepressant-like effects in traditional rodent models of depression. Systemic administration of deprenyl (1.5-12.0 mg/kg IP) shifted choice behavior, significantly increasing markers of PROG lever pressing at a moderate dose (6.0 mg/kg), and decreasing chow intake at 6.0 and 12.0 mg/kg. Intracranial injections of deprenyl into nucleus accumbens (2.0 and 4.0 μg) also increased PROG lever pressing and decreased chow intake. Microdialysis studies showed that the dose of deprenyl that was effective at increasing PROG lever pressing (6.0 mg/kg) also significantly elevated extracellular dopamine in nucleus accumbens. Thus, similar to the well-known antidepressant bupropion, deprenyl is capable of increasing selection of high-effort PROG lever pressing at doses that increase extracellular dopamine in nucleus accumbens. These studies have implications for the potential use of MAO-B inhibitors as treatments for the motivational symptoms of depression and Parkinsonism., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this research was to develop and validate a headspace-solid phase micro-extraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) method for the determination of volatile emerging contaminants in fruit. The method showed good precision (RSD ≤ 14%) and satisfactory recoveries (99.1-101.7%) and LOD and LOQ values ranging between 0.011-0.033 μg kg -1 and 0.037-0.098 μg kg -1 , respectively. The method was applied to investigate the content of volatile emerging contaminants in two varieties of melon fruit (Cucumis melo L.) cultivated adjoining high-risk areas. Glycol ethers, BHT, BHA and BTEX (benzene, toluene, ethylbenzene and xylene) were determined in melon fruit pulps for the first time, with different sensitivities depending on sample and variety. Although the amount of the volatile contaminants in the melon samples were in the order of µg kg -1 , the safety of vegetable crops cultivated near risk areas should be more widely considered. The results showed that this accurate and reproducible method can be useful for routine safety control of fruits and vegetables.

The prognosis and outcome of treatment for chronic hepatitis B virus (HBV) infection are predicted by levels of HBV DNA in serum. These levels are composed of relaxed circular DNA (rcDNA) and double stranded linear DNA in viral particles, whereas, HBV DNA in liver tissue also can be covalently closed circular DNA (cccDNA) or integrated into the human genome. The aim of this study was to investigate the quantitative relation between HBV DNA in serum and tissue, its change over time and how these markers relate to serum levels of hepatitis B surface antigen (HBsAg). Serum and liver biopsies taken from 15 patients with chronic HBV infection on two occasions during 2.7-11.1 years were analyzed retrospectively. At baseline, the median HBV DNA levels in serum were 7.76 log 10  IU/mL in nine hepatitis B e antigen (HBeAg) positive and 3.65 log 10 IU/mL in six HBeAg-negative patients. At follow-up, serum HBV DNA, serum HBsAg, and intrahepatic HBV DNA (ihDNA) levels had declined by 4.36, 0.52, and 1.47 log 10 units, respectively, in seven patients that lost HBeAg, whereas the corresponding reductions were 0.36, 0.30, and 0.39 log 10 units in eight patients with unchanged HBeAg status. We conclude that HBV DNA in liver tissue declined almost 1000 times less than HBV DNA in serum during and after loss of HBeAg. This finding raises the possibility that integrated sequences constitute a significant part of the ihDNA. Alternatively, the greater decline of HBV DNA in serum might be due to yet unknown mechanisms acting downstream of reverse transcription., (© 2017 Wiley Periodicals, Inc.)

In this paper we show the development and validation of a volumetric absorptive microsampling (VAMS™)-LC-MS/MS method for the simultaneous quantification of four antibiotics: piperacillin-tazobactam, meropenem, linezolid and ceftazidime in 10μL human blood. The novel VAMS-LC-MS/MS method has been compared with a dried blood spot (DBS)-based method in terms of impact of hematocrit (HCT) on accuracy, reproducibility, recovery and matrix effect. Antibiotics were extracted from VAMS and DBS by protein precipitation with methanol after a re-hydration step at 37°C for 10min. LC-MS/MS was carried out on a Thermo Scientific™ TSQ Quantum™ Access MAX triple quadrupole coupled to an Accela ™UHPLC system. The VAMS-LC-MS/MS method is selective, precise and reproducible. In contrast to DBS, it allows an accurate quantification without any HCT influence. It has been applied to samples derived from pediatric patients under therapy. VAMS is a valid alternative sampling strategy for the quantification of antibiotics and is valuable in support of clinical PK/PD studies and consequently therapeutic drug monitoring (TDM) in pediatrics., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Hodgkin Lymphoma (HL) is a tumor of B-cell origin characterized by Hodgkin and Reed-Stenberg (H/RS) cells embedded in an inflammatory tissue where numerous cytokines/chemokines contribute to shape the microenvironment, leading to the typical clinical symptoms. We investigated: i) the expression of Interleukin-IL-31 (IL-31) and Thymic Stromal Lymphopoietin (TSLP), two Th2-related cytokines with tumor-promoting and pruritogenic functions, and of the respective receptors in HL invaded lymph nodes by flow cytometry, and ii) the potential association of IL-31/TSLP plasma concentrations with clinical characteristics by ELISA. H/RS cells and the major immune cell types infiltrating HL lymph nodes expressed intracytoplasmic and surface IL-31/TSLP, and their receptors. A subgroup of patients showing at diagnosis elevated IL-31 and TSLP plasma levels had an International Prognostic Score>2, indicative of high risk of relapse, and a subsequent positive interim PET-scan, indicative of insufficient response to chemotherapy. No correlation was found between IL-31/TSLP plasma levels and overall or event-free survival. In conclusion, IL-31/TSLP and their receptors are expressed in HL cells and in immune cells infiltrating affected lymph nodes, where both cytokines may contribute to local immune suppression. The clinical impact of IL-31 and TSLP plasma levels has to be further defined in larger patient cohorts., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no competing financial interests in relation to the work described.

Protein induced by vitamin K absence-II (PIVKA-II) is a potential screening marker for hepatocellular carcinoma (HCC). Limited data are available about its utility in discriminating neoplastic from regenerative nodules at ultrasonography (US) evaluation in cirrhotic patients. Aim of this study was to investigate the diagnostic utility of PIVKA-II in cases showing liver nodules of uncertain diagnosis at US.Ninety cirrhotics with US evidence of liver nodule(s) were enrolled. All patients underwent blood sampling within 1 week of US and were thereafter followed up. HCC was confirmed in 40/90 cases, and in all cases it was in a very early/early stage. All sera were tested for PIVKA-II and alpha-fetoprotein (AFP) at the end of follow-up. PIVKA-II at a cut off of 60 mAU/mL was significantly associated with HCC at both univariate and multivariate analysis (P = .016 and P = .032, respectively). AFP at a cut off of 6.5 ng/mL was not associated with HCC at univariate analysis (P = .246). ROC curves showed that PIVKA-II had 60% sensitivity, 88% specificity, 80% positive predictive value (PPV), and 73% negative predictive value (NPV), whereas AFP had 67% sensitivity, 68% specificity, 63% PPV, and 72% NPV. AUROC curves showed that the combination of both biomarkers increased the diagnostic accuracy for HCC (AUC 0.76; sensitivity 70%, specificity 94%, PPV 91%, and NPV 79%).In conclusion, PIVKA-II is a useful tool for the diagnostic definition of US-detected liver nodules in cirrhotic patients, and it provides high diagnostic accuracy for HCC when combined with AFP.